Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success

Abstract

Source: Gulla, S. et al. Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis. J. Vis. Exp. (2019)

In this video, we demonstrate the separation of bacterial PCR-amplified DNA using agarose gel electrophoresis. Agarose gel functions as a molecular sieve, enabling the negatively-charged DNA to migrate based upon their size under an applied electric field.

Protocol

1. PCR Amplicon Confirmation by Gel Electrophoresis

1. According to the manufacturer's recommendations, prepare a volume of 1.5% (w/v) agarose gel in 1x tris-borate-EDTA (TBE) buffer appropriate for the number of PCR reactions to be tested. Prior to casting, add 5 µL of fluorescent nucleic acid dye per 50 µL of gel solution and mix. Use trays and combs as appropriate for casting, leaving an appropriate number of wells free for DNA reference ladders.
2. After setting, submerge the gel in 1x TBE-buffer in a GE system. Mix 5 µL of PCR product together with 2 µL of loading dye and transfer to gel wells. Add 5 µL of....

Results

Figure 1: Gel electrophoresis verifying the presence of multiple PCR products. The image confirms the presence of multiple PCR amplicons in all 12 lanes containing samples, with the first lane representing the DNA ladder used. The sizes of selected ladder fragments have been indicated,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose, universal, peqGOLD	VWR	732-2789P/732-2788
Tris-borate-EDTA (TBE) buffer	NA	NA	Standard recipe; produced in-house.
DNA Gel Loading Dye (6X)	Thermo Fisher Scientific	R0611
Gel electrophoresis system	As preferred	NA
GelRed Nucleic Acid Stain, 10,000X in water	Biotium	41003	Fluorescent nucleic acid dye 
GeneRuler 50 bp DNA Ladder, ready-to-use	Thermo Fisher Scientific	SM0373	DNA ladder 
UV-based gel imaging/visualisation system	As preferred	NA